High-activity lipase and method of preparation thereof



United States Patent 3,189,529 HIGH-ACTIVITYLIPASE AND METHOD 0F PREPARATION THEREOF Koichi Yamada, Tokyo, and Haruo Machida, Saitamaken, Japan, assignors to Meito Sangyo Kabushiki Kaislm, Nishi-lru, Japan, a corporation of Japan No Drawing. Filed Sept. 24, 1962, Ser. No. 225,908 Claims priority, application Japan, Sept. 25, 1961, 3634.236 6 Claims. (Cl. 195-62) This invention relates to a high-activity lipase obtained by the cultivation of Candida cylindracea, a new species belonging to the genus Candida, and to the method of preparing the same. More particularly, it-relates to a high-activity lipase which 'is ohtainedby cultivating in a; suitable medium Candida cylindrac'ea' having A.T.C.C.

3,189,529 Patented June 15, 1965 No. 14,830. Its microbiological characteristics are as follows:

(1) Cell in malt extract: After 3 days at C. the cells are long-oval to cylindrical or pseudomycelical, (0.8-3.5) x (LO-21);, single, in pseudomycelium.

(2) Slide cultures on potato agar:.Well developed pseudomycelium is formed.

. (3) Pellicle formation: Grey, slightly rough and creeping truefilm is formed on the malt medium. (4) Sporulation: No formation.

(5) Streak culture: Greyish-white, umbonate at the elevation, undulate at the margin, very moist, dull, wrin kle d for netted-form at the surface.

(6) Fermentation: Glucose+ galactose-sucnose-l- (very weak) maltoselactoseraffinose-..

(7) Sugar assimilation: Glucose+ galactose+ sucrose-k maltose+ (very weak) lactosexylose,+.

(8) KNO assimilation: Negative.

(9) Growth in ethanol medium: A pellicle is formed. i v

(10) Splitting of arbutin: Negative.

The differences between this yeast, Candida cylindracea, and Candida solqni and Mycotorula lipolytica ,are as shown in Table I, below. Thus, it is definite that'this is a new species. 1

TABLE I Mycoiorula lipolytica Candida solani Candida culindracen pairs, or in chains as (a-4.5) x (4-2: Smooth yellowish L.

Smooth yellowish t (very weak) wrinkled, netted greyishwhite.

+. Y (very weak).

+. (very weak).

, Splitting oft irliiitlnii:

(P v (very weak) Sui-rost- Absent or slightly positive.

tained was low,and thus itwasimpossible to prepare a high-activity lipase using these yeasts.

When research wasconductedfor the purpose of obtaining a lipase having sufiiciently high activity, numerous microbes from air, soil, sewage, etc., were isolated. -An

investigation into their productswas made and a new species belonging to the genus Candida was discovered. The production of lipase in the Candida culture medium was remarkably high. Hence, an easy and low cost method of preparing a lipase of very high activitywas established.

Accordingly, it is an object of the present invention to provide a high-activity lipase produced by'a new yeast species and the method of preparing the same.

Another object of the invention is to provide for medicinal purposes'a high-activity lipase possessing very favorable activity maintenance and preservability as well as excellent acid stability, and the method of preparation thereof.

Other objects and advantages of this invention will be apparent from the following description.

The lipase-producing yeast used in the present invention is a yeastthat was isolated from the soiland is a strain belonging to a new species which we named Candida cylindra cea nov. sp. and which has A.T.C.C.

The characteristics of this yeast are not restricted to those mentioned hereinabove, as it is capable of being changed artificially .or naturally. Moreover, as long as they possess the ability at least of producing lipase, the variants and mutants of this yeast can also be used in the rnethod of this invention. I

Accordingly, the yeast species used in the present invention include Candida cylindracea and the variants and I mutants thereof that possess most of the distinctive properties of the parent yeast.

The method of preparing lipase according to the present invention is described hereinafter.

The collection or preparation as used in this invention means the acquisition of lipase in a state in which it is more readily purified, or in a higher concentration or higher purity.

.dl'qb'a and the variants and mutants thereof that possess most of the distinctive properties of the parent yeast. The

carbon source used may be, for example, sucrose, lactose,

perature for growth is selected, normally -35 C., and The pH of'themedium is suitably'from 5.0 to 8.0, and normally the preferably -33 C. being-suitable.

cultivation is carried out for l-7 days. Although the cultivationmay be-carried out by means of either solid or liquid culture, generally more favorable results are obtained by liquid culture. Preferably the culture is carried out under aerobic conditions. Thus, while in the case of liquid culture, surface culture may be employed, better results are obtained by means of shaking culture or submerged culture.

As a result of cultivation, as described above, highactivity lipase is produced inv the medium. Collecting the high-activity lipase from the culture medium, in the case of solid culture, is accomplished by extracting with water or an aqueous solution, while in the case of liquid culture it is done by removal of the cell, whereby in both cases a high-activity lipase-containing solution ,can be collected.' The aqueous solution may be one containing salts such as sodium chloride, potassium chloride, etc., or a solution containing acids such as hydrochloric acid, citric acid, acetic acid, etc., as well as one of the numerous buffer solutions. 1 v

The enzyme solution containing high-activity lipase can then be concen-trated or solidified by carrying out a purification treatment such as precipitation with organic solvents, salting out, concentration under reduced pressure, purification by means of ion exchangers, .etc. The organic solvents used in this case include the water miscible organic solvents such as methanol, ethanol, isopropanol, acetone, etc. When the foregoing solvents are used, the concentration at which the organic solvents are added is such that at 20% by volume the enzyme remains dissolved but at 60% by volume the collection of a precipitating portion will result. As the salting out the invention is not to be limited thereby, it being possible to make various changes without departing fromthe spirit and scope of the invention.

Example I A 50 ml. portion of a medium whose pHis 7.2 and which contains 2% of starch, 2% of soybean flour, 0.1%

of ammonium sulfate, 0.5% of potassium diphosphate agent, anyof the salts that dissolve readily in water may i be used, such as ammonium sulfate, sodium sulfate, magnesium sulfate, etc. The concentration at which these suits are added is, for example, in the case of; ammonium sulfate, such that at 10% saturation the enzyme remains dissolved, but at 30% saturation the collection of a precipitating portion 'will result. ,Purification by means of an ion exchange resin can also be utilized.

The high-activity lipase that is provided by the present invention can remain in storage even at room temperatures over an extended period of time without any lowering of its activity, if stored after drying. The optimum pH of this enzyme is in the neighborhod of 7, and it is extremely stable between pH 5.0 and 8.5. Even at pH 3.0, it is acid stable to a considerable extent, only of its activity being inactivated upon being exposed for 30 minutes to a.temperature of 30 C. This property of the lipase is especially advantageous when it is used for medicinal purposes.

The high-activity lipase of the invention, which is capable of being economically and very advantageously mass-produced continuously on a commercial basis, can

be utilized not only for medicinal purposes but also for other uses such as detergent use, cosmetic use, food additive use, etc.

In order to more clearly understand the present invention, examples illustrating the mode of practicing the invention are given below. It will be understood that the examples are merely intended in an illustrative sense, and

and 0.1% of magnesium sulfate is poured into a shaking flask of 500 ml. capacity. The solution is sterilized for 5 minutes at 0., and then is inoculated with Candida cylindracea, after which shaking culture is carried out at 30 C. The cell is removed byeentrifuge 72 hours later. When ammonium sulfate is gradually added tothe supernatant and its degree of, saturation. is brought to 30%,

the lipase in the liquid precipitates'and the precipitate is collected by centrifuge. By drying under reduced pressure at room temperature, a powder of lipase is obtained. The yield is about 2% based on the volume of the medium. The activity of this powdered lipasemeasured by means of hydrolysing of fat is as follows:

Rate of hydrolysing of fat, percent Present powdered lipase (unpurified), 5 mg. .4 26 Lipase powder (purified) produced by Mann Research Laboratories, 5 mg. .25

The foregoing test was performed by the following method of Nord et al. Namely, 5 ml. of an emulsion of olive oil, 3-ml. of a buffer solution and 1 ml. of the enzyme solution are incubated at 37 C. for 4 hours. The rate of hydrolysing of fat ismeasured by means of titration I of the fatty acid with a caustic soda solution.

Example II a A 50 ml. portion of a medium whose pH is 7.2 and which contains 2% of xylose, 2% of soybean flour, 0.1% of ammonium sulfate, 0.1% of magnesium sulfate and 0.5% of potassium diphosphate is poured into a shaking flask of 500 ml. capacity. After sterilizing for 5 minutes at 115 C., the solution is inoculated with thel'Cmidida. cylindracea, and then shaking culture is carried out at 30C. The cell is removed by centrifuge 72 hours later. When ethanol is added gradually to the supernatant .until the concentration thereof is brought to 60% by volume, all of the lipase in the liquid precipitates. This precipitate is collected and dried under reduced pressure at room temperature whereby powdered lipase is obtained. The

yield of this powdered lipase is about 2% based on the volume of the medium. This powdered lipase even though stored for more than a month at room-temperature does not lose its activity, which interrns of hydrolysing offaitis25%.

Having thus described the nature of the .invention, what we claimis: l

1. A lipase having high-activity at a pH from 3.0-8.5 obtained by cultivating at a temperature of from 20-35 C. in a suitable medium Candida cylindracea A.T.C.C. No. 14,830.

2. A method of preparing high activity lipase which comprises cultivating ina suitable medium Candida cylindracea A.T.C.C. No. 14,830, wherein lipase is produced, and thereafter collecting the lipase from the culture. I

3. The method acording to claim 2 in which said medium contains at least a carbon source, a nitrogen source cylfndracea A.T.C.C. No. 14,830 wherein lipase is pro 7 5 duced, and thereafter collecting lipase from the culture obtained.

6. The method according to claim 5 wherein the lipase is collected from the culture by a step selected from the References Cited by the Examiner Journal'of Food Science, 26 (No. 5), pp. $1842 4 (1961 reprint of l95-62 lipase.

Cook: The Chemistry and Biology of Yeasts," 1958,

group consisting of (l) precipitating with an organic 501- 5 52, 55, A d mic Pre Inc New York,

A. LOUIS MONACELL, Primary Examiner. A L RAHAM H. WINKELSTEIN, Examiner.

vent, (2) salting out, (3) concentrating under reduced pressure, (4) purifying with an ion exchanger and (5) a combination of said steps. 

1. A LIPASE HAVING HIGH-ACTIVITY AT A PH FROM 3.0-8.5 OBTAINED BY CULTIVATING AT A TEMPERATURE OF FROM 20-35* C. IN A SUITABLE MEDIUM CANDIDA CYLINDRACEA A.T.C.C. NO. 14,830. 